Interferon receptor binding peptides

ABSTRACT

A polypeptide for use as an interferon receptor-binding peptide, said polypeptide selected from the group of peptides having an amino acid sequence substantially of the formulae; CYS-LEU-LYS-ASP-ARG-HIS-ASP; ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-LEU-ASN-ASP; ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS; TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA; TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA; TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR; and GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP. The polypeptides are useful for delivering a pharmaceutically active drug to a cell.

This application is a 371 of PCT/CA93/00279 filed Jul. 06, 1993 which isa continuation of Ser. No. 07/980,525 filed Nov. 20, 1992, nowabandoned, which is a continuation of Ser. No. 07/909,739, filed Jul. 7,1992, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to receptor binding domains in proteinsand more specifically, to specific peptides that interact with the Type1 human interferon receptor complex.

In order for any pharmaceutical composition to be therapeuticallyeffective, it must be formulated in such a way that it reaches thedesired target cells intact. Moreover, once at the site of action, thetherapeutic must specifically interact with the target cells. Thus, thedesign and development of suitable carrier molecules, that maythemselves be inert or active, allows for effective targeting ofclinically active drugs. Much work has been done in the field ofcarriers for pharmaceutical compositions. Most recently, peptides havebeen identified as potentially suitable carriers for pharmaceuticalcompositions.

The interferons (hereinafter referred to as IFNs) are a family ofbiologically active proteins that are classified into three majorgroups, namely, IFN-alpha, IFN-beta and IFN-gamma. IFNs affect a widevariety of cellular functions, related to cell growth control, theregulation of immune responses and more specifically, the induction ofantiviral responses. The ability of IFNs to modulate cell growth isobserved with many cell types and is particularly effective in the caseof tumor cells, which has led to the widespread interest in the use ofIFNs for the treatment of neoplaslias.

The presence of a specific receptor at the cell surface is the firstrequirement for IFN action. Cells that lack these specific receptors areresistant to the effects of IFN. Receptor binding studies haveidentified the existence of at least two functional IFN receptors thatare integral parts of the cell membrane on human cells. Branca, A. A.and Baglioni, C., (1981) Nature 294, 768-770 report that IFN-alpha andIFN-beta bind to one type of receptor and Anderson, P. et al, (1982) J.Biol. Chem. 257, 11301-11304 report that IFN-gamma binds to a separatereceptor. IFN receptors are ubiquitous and more specifically, areupregulated in metabolically active cells such as cancer cells andinfected tissues. Although several of the effects of IFNs such as theantiviral state, take several hours to develop, signal transductionimmediately following the binding of IFN to its receptor is a rapidevent. Since metabolic changes, such as increases in the transcriptionalrate of some IFN-induced genes can be detected within five minutes ofthe addition of IFN, at least some of the transmembrane signals must bevery rapid. Hannigan et al, (1986) EMBO J. 5, 1607-1613 suggest thatreceptor occupancy modulates the transcriptional response of specificgenes to IFN. Indeed, there is accumulating evidence to suggest thatthere is a direct relationship between the number of receptors occupiedand the amount of signal that is transduced to the cell nucleus. Thesetransduced signals result in altered gene expression in the nucleus,which mediates the subsequent biological responses.

Extensive studies were undertaken to define those critical clusters ofamino acids in the different IFN-alphas and IFN-beta that interact withthe Type 1 IFN receptor complex. It is thought that these criticalpeptide domains would serve as prototypes for synthetic peptides thatare useful as carriers for pharmaceutical compositions.

SUMMARY OF THE INVENTION

Thus, the present invention is directed to novel peptides which arecarriers for pharmaceutical compositions.

More specifically, the invention is directed to novel IFN-receptorbinding peptides that are designed as carriers for pharmaceuticalcompositions.

To this end, in one of its aspects, this invention provides a novelpeptide having an amino acid sequence of CYS-LEU-LYS-ASP-ARG-HIS-ASP.(SEQ. ID NO. 1)

In another of its aspects, the invention provides a novel peptide havingan amino acid sequence ofASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP.(SEQ. ID NO. 2)

In still another of its aspects, the invention provides a novel peptidehaving a sequence of amino acids as follows:ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS.(SEQ. ID NO. 3)

In another of its aspects, the invention provides a novel peptide havingan amino acid sequence of: TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA.(SEQ. ID NO. 4)

The invention also provides a novel peptide having an amino acidsequence of:TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA.(SEQ. ID NO. 5)

A further aspect of the invention is the provision of a novel peptidehaving an amino acid sequence of: TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR. (SEQ.ID NO. 6)

A still further aspect of the invention is the provision of a novelpeptide having an amino acid sequence of:GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP. (SEQ. ID NO. 7)

In yet another of its aspects, the invention provides a pharmaceuticalcomposition which comprises an active drug and a suitable carrier, thecarrier having been selected from the group of peptides having an aminoacid sequence of CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO. 1);ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP(SEQ. ID NO. 2);ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS(SEQ. ID NO. 3); TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA (SEQ. IDNO. 4);TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA(SEQ. ID NO. 5); TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR (SEQ. ID NO. 6); andGLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP (SEQ. ID NO. 7).

The invention also provides a pharmaceutical composition which comprisesan active drug and a suitable carrier, the carrier having been selectedfrom the group of peptides substantially of the formula:CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO. 1);ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP(SEQ. ID NO. 2);ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-TYR-ALA-ASN-VAL-VAL-HIS-GLN-ILE-ASN-HIS(SEQ. ID NO. 3); TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA (SEQ. IDNO. 4);TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA(SEQ. ID NO. 5); TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR (SEQ. ID NO. 6); andGLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP (SEQ. ID NO. 7).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the growth inhibitory activities of variantIFN-alphas in T98G cells.

FIG. 2 shows five charts illustrating receptor binding characteristicsof variant IFN-alphas on T98G cells.

FIG. 3 shows four charts illustrating receptor binding characteristicsof variant IFN-alphas on T98G cells.

FIG. 4 shows secondary structure characteristics of different IFN-alphaspecies according to amino acid sequence analyses.

FIG. 5 is a representation of a model for the tertiary structure of Type1 IFNs.

FIGURE LEGENDS

FIG. 1

Growth inhibitory activities of variant IFN-αs in T98G cells.

Cells were incubated with the different IFN-α species, at the indicateddoses, at 37° C. for 96 hr, then growth inhibition was estimated byspectrophotometric determination, as described.

Values represent the average of triplicate determinations and exhibiteda SE of ±4%. □ IFN-α2a; ▪ (4-155)IFN-α2a; Δ 4-155(S98)IFN-α2a; ▴4-155(L98)IFN-α2a; ⋄ (ESML)IFN-α2a; ♦ (A30,32,33)IFN-α2a

FIG. 2

Receptor binding characteristics of variant IFN-αs on T98G cells.

Binding isotherms. 3.5×10⁵ T98G cells were incubated for 2 hr at +4° C.with the indicated concentrations of ¹²⁵ I-IFN-αCon₁, (A), ¹²⁵I-4-155(S98)IFN-α2a, (B), or ¹²⁵ I-IFN-α1Nδ4, (C). Inset into A, B and Care the corresponding Scatchard plots.

Competitive displacement profiles. 3.5×10⁵ T98G cells were incubated at+4° C. for 2 hr with 10 ng/ml ¹²⁵ I-IFN-αCon₁, (D), 3.7 ng/ml ¹²⁵I-4-155(S98)IFN-α2a, (E), or 300 ng/ml ¹²⁵ I-IFN-α1Nδ4, (F), containingno unlabeled competitor (100% bound) or the indicated concentrations ofIFNs.

For D and F: ▪ IFN-αCon₁ ; □ IFN-α1Nδ4.

For E: ▪ IFN-α2a; □ 4-155(S98)IFN-α2a; Δ 4-155(L98)IFN-α2a.

The values shown were obtained by subtracting non-specific counts/minbound from total counts/min bound. Non-specific binding was determinedin the presence of a 100-fold excess of unlabeled IFN. The pointsrepresent the mean of triplicate cultures and exhibited a S.E. or ±3%.

FIG. 3

Receptor binding characteristics of variant IFN-αs on T98G cells.

Binding isotherms

3.5×10⁵ T98G cells were incubated for 2 hr at +4° C. with the indicatedconcentrations of ¹²⁵ I-(4-155)IFN-α2a, (A), and ¹²⁵I-4-155(L98)IFN-α2a, (B). Inset into A and B are the correspondingScatchard plots.

Competitive displacement profiles

3.5×10⁵ T98G cells were incubated at +4° C. for 2 hr with 20 ng/ml ¹²⁵I-(4-155)IFN-α2a, (C), or 8 ng/ml ¹²⁵ I-4-155(L98)IFN-α2a, (D),containing no unlabeled competitor (100% bound) or the indicatedconcentrations of IFNs.

▪ IFN-α2a; □ (4-155)IFN-α2a; Δ 4-155(L98)IFN-α2a; ▴ (ESML)IFN-α2a; ⋄(A30,32,33)IFN-α2a.

The values shown were obtained by subtracting non-specific counts/minbound from total counts/min bound. Non-specific binding was determinedin the presence of a 100-fold excess of unlabeled IFN. The pointsrepresented the mean of triplicate cultures and exhibited a S.E. of ±3%

FIG. 4

Predicted secondary structure characteristics of different IFN-α speciesaccording to amino acid sequence analyses.

Hydrophilicity, H, and surface probability, S, profiles are depicted foreach of the IFN-αs and IFN-β, whose designations are on the left handside of each pair. Amino acid residue position is indicated along thehorizontal axes of the graphs. The critical domains, comprising residues29-35, 78-95 and 123-140, are boxed.

FIG. 5

Model for the tertiary structure of Type I IFNs.

This model incorporates a helical bundle core, composed of the 5 helicesA-E. The loop structures that constitute the proposed receptorrecognition epitopes, residues 29-35 and 130-140, shown here as heavilyshaded, broad lines, are aligned such that they dock in the receptorgroove as shown. The third region implicated in the active conformationof the Type I IFNs, 78-95, is not buried in the receptor groove and isconfigured to allow binding to its cognate epitope on another Type 1 IFNreceptor. The shaded areas in helices C and D represent residues thatare critical for maintaining the correct structural presentation of thecorresponding contiguous recognition epitopes (see text).

DESCRIPTION OF THE PREFERRED EMBODIMENT

Biologically active proteins have an optimum active configuration thatis composed of discrete and unique strategic domains along thepolypeptide. These critical structural domains determine such parametersas receptor binding and effector functions. Characterization of thesestrategic domains, that includes defining their spatial configurationand effector functions, will clarify the sequence of events comprisingand initiated by receptor binding and that lead to specific biologicalresponses.

For a therapeutic agent to be optimally active, it must be delivered tothe specific site of action intact and must interact with the targettissues. In a number of clinical conditions, such as uncontrolledproliferation in neoplastic tissues, or infected tissues, or inflamedtissues, the cells express abundant Type 1 IFN receptors, that is,IFN-alpha and IFN-beta receptor expression at the cell surface isupregulated. It has been determined that specific peptides are capableof recognizing and binding to these cell surface receptors. Once bound,the ligand-IFN receptor complex is transported into the cell.

The present invention relates therefore to novel carriers which comprisepeptides of specific amino acid sequences. These sequences are:

(i) an amino acid sequence of CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO.1);

(ii) an amino acid sequence ofASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP(SEQ. ID NO. 2);

(iii) an amino acid sequence ofASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS(SEQ. ID. NO. 3);

(iv) an amino acid sequence of:TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA (SEQ. ID NO. 4);

(v) an amino acid sequence of:TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA(SEQ. ID NO. 5);

(vi) an amino acid sequence of: TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR (SEQ. IDNO. 6); and

(vii) an amino acid sequence of: GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP (SEQ.ID NO. 7).

These novel peptide/carriers have been incorporated into interferons toestablish their claimed utility. The following description will be madein conjunction with experiments using interferons having the novelcarriers incorporated therein but the invention is not to be restrictedto such interferons.

Fish et al in J. IFN Res. (1989) 9, 97-114 have identified three regionsin IFN-alpha that contribute toward the active configuration of themolecule. These three regions include: 10-35, 78-107 and 122-166.

The structural homology and symmetry observed among a number ofhaemopoietic cytokine receptors, and specifically the IFN receptors andtissue factor, the membrane receptor for the coagulation protease factorVII, lends support to the functional receptor binding model that wasproposed by Bazan, J. F., Pro. Natl. Acad. Sci. (1990) 87, 6934-6938.This model invokes the presence of a generic binding through that allowsrecognition of conserved structural elements among different cytokines.The present inventor's data supports such a model, at least for thedifferent IFN-alpha molecular species and IFN-beta, since they haveidentified two conserved elements in the Type 1 IFNs that effectreceptor recognition. A third structural element, that is an exposedrecognition epitope, confers specificity of cytokine function, includingspecies specificity.

Experiments were conducted using IFNs shown in Table 1:

                                      TABLE 1                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                     __________________________________________________________________________

Table 1

The foregoing table illustrates the amino acid sequence alignment of thedifferent Type 1 IFNs. The designation of the various IFNs is shown inthe left hand column and the sequence of IFN-beta is aligned with theother IFNs, commencing with residue 4, to achieve the greatest homology.The critical domains comprising residues 29-35, 78-95 and 123-140 areboxed. The letter codes for the amino acids are as follows: A, ala; C,Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M,Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; andY, Tyr.

IFN-alpha2a and the various derivatives were provided by I.C.I.Pharmaceuticals Division of the UK; IFN-alphaCon₁ was supplied by Amgenof the USA and IFN-alpha₁ Nδ4 was supplied by Schering Plough Corp ofthe USA.

IFN-alpha2a, (4-155)IFN-alpha2a, 4-155(S98)IFN-alpha2a and4-155(L98)IFN-alpha2a had specific activities of 2×10⁸ U/mg protein;(A30,32,33)IFN-alpha2a was inactive in antiviral assays and(ESML)IFN-alpha2a had a specific activity of 7.5×10⁶ U/mg protein;IFN-alphaCon₁ had a specific activity of 3.0×10⁹ U/mg protein; andIFN-alpha₁ Nδ4 had a specific activity of 7.1×10⁶ U/mg protein.

The cell culture used comprised T98G cells which were derived from ahuman glioblastoma multiforma tumor and which express in culture anumber of normal and transformed growth characteristics. These cells maybe routinely subcultured as monolayers, in modified minimum essentialmedium (hereinafter referred to as alpha-MEM), and supplemented with 10%(v/v) fetal calf serum (hereinafter referred to as FCS).

An in vitro assay for antiviral activity was conducted. T98G cells wereseeded at a density of 1.5×10⁵ /ml in 200 μl alpha-MEM supplemented with10% FCS in 96-well Microtest (trade mark) II tissues culture plates andtreated with dilutions of the IFN preparations for 24 hours. At the timeof virus innoculation, the IFNs were removed and 10⁴ PFU EMCV was addedto individual wells in 100 μl alpha-MEM, 2% FCS. After 24 hours, thecells were ethanol (95%) fixed and the extent of EMCV infection wasdetermined by spectrophotometric estimation of viral CPE. The fixedcells were crystal violet (0.1% in 2% ethanol) stained and destained(0.5M NaCl in 50% ethanol), and the inhibition of virus infection wasestimated from absorbance measurements at 570 nm using a Microplate(trade mark) Reader MR600 and a calibration of absorbance against cellnumbers. IFN titers were determined using a 50% cytopathic end-point andconverted to international units using an NIH IFN-alpha standard (Ga23-901-527).

An in vitro assay for growth inhibitory activity was conducted. T98Gcells were seeded in 96-well Microtest II tissue culture plates at adensity of 5×10³ /ml and either innoculated with two-fold serialdilutions of different molecular species of IFN-alpha or left untreated.After incubation, at 37° C. for 96 hours, the cells were ethanol fixed(95%), crystal violet (0.1% in 2% ethanol) stained and destained (0.5MNaCl in 50% ethanol), then growth inhibition was estimated fromabsorbance measurements of destained cells at 570 nm (using a MicroplateReader MR600 and a-calibration of absorbance against cell numbers).

The results of these experiments are shown in FIG. 1. The valuesrepresented are the average of triplicate determinations and exhibited aSE of +/-4%. Whereas IFN-alpha2a, (4-155)IFN-alpha2a,4-155(S98)IFN-alpha2a and 4-155(L98)IFN-alpha2a demonstrate comparablegrowth inhibitory activities within the error of the assay,(ESML)IFN-alpha2a and (A30,32,33)IFN-alpha2a do not exhibitantiproliferative activity. Similarly, IFN-alpha₁ Nδ4 has minimalantiviral activity (7.1×10⁶ U/mg protein) and no demonstrableantiproliferative activity over the dose range examined.

The next series of experiments examined IFN-receptor interactions.Labelling was carrier out using ¹²⁵ I using a solid phaselactoperoxidase method. A 100 μl reaction mixture containing 10 μl 3%B-D-glucose, 10 μl hydrated Enzymo-beads (trade mark) (available fromBioRad in California, USA) 2 μCi Na¹²⁵ I and 20 μg HuIFN-alpha in PBS,pH 7.2, was reacted overnight at +4° C. Free ¹²⁵ I was separated fromIFN-bound ¹²⁵ I on a 12 ml Sephadex (trade mark) G-75 column,equilibrated in PBS containing 1 mg/ml BSA. Iodination caused nodetectable loss of antiviral activity. Fractions containing maximumantiviral activity were pooled and contained 95% TCA (10%) precipitableradioactivity.

Sub-confluent cell monolayers were incubated at +4° C. in alpha-MEMcontaining 2% FCS and indicated concentrations of ¹²⁵ I-IFN-alpha. After2 hours, the binding medium was aspirated and the cultures were washedtwice with ice-cold PBS. The cells were solubilized in 0.5M NaOH andradioactivity counted in a Beckman (trade mark) 5500 *-counter.Specificity of binding was determined in parallel binding assayscontaining a 100-fold excess of unlabeled growth factor. For competitiveexperiments, specified amounts of unlabeled competitor were included inthe reaction mixture together with radiolabelled ligand.

Specific ¹²⁵ I-IFN-alpha binding data were used to determine receptornumbers and dissociation constants, K_(d). With increasingconcentrations of ¹²⁵ I-ligand in the cellular binding reactions,respective specific binding activities corresponding to each ¹²⁵I-ligand concentration was calculated.

In FIG. 2, panel A illustrates the results using ¹²⁵ I-IFN-alphaCon₁ ;panel B illustrates the results using ¹²⁵ I-4-155(S98)IFN-alpha2a; andpanel C illustrates the results using ¹²⁵ I-IFN-alpha₁ Nδ4. Inset intopanels A, B and C are the corresponding Scatchard plots. The competitivedisplacement profiles are shown in panels D, E and F using 10 ng/ml of¹²⁵ I-IFN-alphaCon₁, 3.7 ng/ml of ¹²⁵ I-4-155(S98)IFN-alpha2a and 300ng/ml of ¹²⁵ I-IFN-alpha₁ Nδ4 respectively, with no unlabeled competitor(100% bound) or the indicated concentrations of IFNs. The values shownwere obtained by subtracting non-specific counts/min bound from totalcounts/min bound. Non-specific binding was determined in the presence ofa 100-fold excess of unlabeled IFN. The points represent the mean oftriplicate cultures and exhibited a S.E. of +/-3%.

In FIG. 3, panel A illustrates the results using ¹²⁵I-(4-155)IFN-alpha2a and panel B illustrates the results using ¹²⁵I-4-155(L98)IFN-alpha2a. Inset into panels A and B are the correspondingScatchard plots. The competitive displacement profiles are shown inpanels C and D using 20 ng/ml of ¹²⁵ I-(4-155)IFN-alpha2a and 8 ng/ml of¹²⁵ I-4-155(L98)IFN-alpha2a, with no unlabeled competitor (100% bound)or the indicated concentrations of IFNs. The values shown were obtainedby subtracting non-specific counts/min bound from total counts/minbound. Non-specific binding was determined in the presence of a 100-foldexcess of unlabeled IFN. The points represent the mean of triplicatecultures and exhibited a S.E. of +/-3%.

FIGS. 2 and 3 illustrate the steady state receptor bindingcharacteristics of the different IFN-alpha molecular species on T98Gcells at +4° C. Specific binding to sub-confluent T98G monolayers isresolved into a biphasic Scatchard plot. This IFN binding heterogeneityhas been shown to result from negatively cooperative site-siteinteractions between the ligand receptors. Analysis of the IFN-alpha2abinding data reveals both high and low affinity binding components, withK_(d) s of 2-3×10⁻¹¹ M and 2-5×10⁻⁹ M, respectively. It was found that¹²⁵ I(ESML) IFN-alpha2a exhibited no detectable binding activity onproliferating (log phase) T98G cells at +4° C. ¹²⁵ I-IFN-alphaCon₁binding to cells was resolved into high affinity K_(d) 7.7×10⁻¹² M) andlow affinity (K_(d) 1.4×10⁻⁹ M) components as shown in FIG. 2A.Similarly, ¹²⁵ I-4-155(S98)IFN-alpha2a (FIG. 2B), ¹²⁵I(4-155)IFN-alpha2a (FIG. 3A) and ¹²⁵ I-4-155(L98)IFN-alpha2a (FIG. 3B)exhibited binding heterogeneity on T98G cells, with high and lowaffinity components comparable to IFN-alpha2a. ¹²⁵ I-IFN-alpha₁ Nδ4binding to T98G cells was resolved into a monophasic Scatchard plot,with a single low affinity binding component of K_(d) 10⁻⁷ M (FIG. 2C).Indeed, competitive binding studies with either ¹²⁵ I-IFN-alphaCon₁(FIG. 2D) or ¹²⁵ I-IFN-alpha₁ Nδ4 (FIG. 2F), confirmed that IFN-alpha₁Nδ4 has a weaker affinity for the IFN-alpha receptor on T98G cells thanIFN-alphaCon₁. Substitution of the cysteine residue at position 98 inIFN-alpha2a with a serine, does not affect the polarity or chargedistribution of the side chain at this position (CH₂ --SH to CH₂ --OH),yet substitution with a leucine residue does introduce an aliphatic sidechain and hence alter the polarity (CH₂ --SH to CH--(CH₃)₂). Thisalteration in side chain polarity at this residue position is notreflected in altered affinity characteristics for the IFN-alpha receptor(FIG. 3B). As would be anticipated, substitution of the cysteine residueat position 98 with serine, did not affect receptor bindingcharacteristics (FIGS. 2B,E). The data from the competitive bindingstudies, indicate that the IFN-alpha2a variants (ESML)IFN-alpha2a and(A30,32,33)IFN-alpha2a, are unable to bind to the IFN-alpha receptor(FIGS. 3C,D).

Since the amino acid sequence dictates the native conformation of aprotein, the inventor has ascribed protein structure for the differentIFN-alphas and IFN-beta. Receptor recognition epitopes arecharacteristically hydrophilic and located on the surface of the bindingmolecule. Generally, sites for molecular recognition in proteins arelocated in loops or turns, whereas alpha-helices are involved inmaintaining the structural integrity of the protein. Close examinationof the hydrophilicity and surface probability plots of IFN-alpha2a showsthat, in those regions that are critical for the active configuration ofIFN-alpha, namely 10-35, 78-107 and 123-166, altering the cysteine at 98has no effect on these determinants (FIG. 4), and indeed, does notaffect biological activity (FIG. 1).

FIG. 4 illustrates predicted secondary structure characteristics ofdifferent IFN-alpha species according to amino acid sequence analyses.Hydrophilicity (H) and surface probability (S) profiles are depicted foreach of the IFN-alphas and IFN-beta whose designations are on the lefthand side of each pair. Amino acid residue position is indicated alongthe horizontal axes of the graphs. The critical domains comprisingresidues 29-35, 78-95 and 123-140 are boxed.

In IFN-alpha2a, in the carboxy-terminal domain there are essentially 3hydrophilic residue clusters that are likely located on the surface ofthe molecule (FIG. 4). Deletion of the cluster closest to thecarboxy-terminus, in (4-155)IFN-alpha2a, has no effect on antiviralspecific activity, growth inhibitory activity (FIG. 1), or receptorbinding characteristics (FIG. 3), compared with the full lengthIFN-alpha2a. Thus, for receptor recognition, the region 155-166 does notinfluence the active configuration of the previously defined strategicdomain 123-166. Interestingly, there are two peaks of hydrophilicity inthis carboxy-terminal region, that spans residues 123-140, thatcorrespond to a helical bundle and loop structure. In the human, equine,bovine, ovine, rat and murine IFN-alphas, human and murine IFN-beta, cowtrophoblast IFN (TP-1) and horse IFN-omega, all designated Type 1 IFNs,these structural motifs are highly conserved (FIG. 4), lending credenceto the notion that this carboxy-terminally located domain is criticalfor receptor recognition for the Type 1 IFNs. The alpha-helicalstructure, that constitutes residues 123-129, allows the appropriatepresentation of the loop structure around residues 130-140, and thisloop structure serves as a recognition epitope for receptor binding.This conclusion is consistent with reports that the region thatcomprises residues 123-136 influences biological activities on human andmurine cells. Further examination of the 10-35 domain, reveals a singleregion that is likely located on the surface of the molecule andcontains hydrophilic residues, namely 29-35. Other reports haveimplicated the amino-terminal region of IFN-alpha, in particular aminoacid residue 33, as critical for biological activity on human and bovinecells. The IFN-alpha2a variants (A30-32,33)IFN-alpha2a and(E5,S27,M31,L59)IFN-alpha2a, that have lost biological activity andreceptor binding characteristics, no longer present this cluster ofresidues near the surface of the molecule, (FIG. 4). This regionconstitutes a loop structure. In IFN-alpha₁ Nδ4, the amino acid residuesthat immediately precede the critical 29-35 cluster are different tothose in IFN-alpha2a, and thus affect the presentation of this receptorbinding epitope somewhat, according to the different predictivealgorithms the inventor has employed. The data in FIG. 4 suggest thatthe cluster of hydrophilic residues that do constitute this receptorrecognition epitope will be located near the surface of the molecule inIFN-alpha₁ Nδ4. However, substitution of the lysine residue at position31 by a methionine residue, affects the configuration of this receptorrecognition epitope, thereby affecting the biological effectiveness ofIFN-alpha₁ Nδ4. In the human and murine IFNs, the loop structure thatincludes residues 29-35, is conserved, yet CLKDRHD is presented asCLKDRMN and NLTYRAD, respectively (see FIG. 3). In murine consensusIFN-alpha, MuIFN-alphaCon, this epitope is conserved as CLKDRKD, where H(histidine) to K (lysine) is a conservative change with respect to sidechain group and charge. Considerable sequence homology with the humanresidues 29-35 is also apparent among the murine, equine, ovine, bovineand rat IFN-alphas, as well as for cow TP-1 and horse IFN-omega. TheType 1 IFNs share conserved receptor recognition epitopes in the 29-35and 123-140 regions. Some variance is seen in the human and murineIFN-beta in the 29-35 region, although the presentation of this epitopeas a loop structure is conserved.

The third strategic region with respect to the active configuration ofIFN-alpha spans residues 78-107. A hydrophilic cluster of amino acidresidues that are likely located on the surface constitute residues83-95 (FIG. 4). These residues probably present as a contiguous helicalbundle and a loop structure. Several amino acid residues around position78 also appear to be located at the surface as part of the helicalbundle. The inventor has shown that substitution of the cysteine atposition 98 with either a serine (S) or a leucine (L) does not affectthe receptor binding characteristics of IFN-alpha2a, hence the inventorinfers that those residues beyond 95, in the previously defined domain78-107, are likely not critical for receptor recognition in IFN-alpha,since they appear not to be located at the surface of the molecule. Thealpha-helical structure allows the appropriate presentation of therecognition epitope that comprises residues 88-95. Of note is thevariance in this region between the human IFN-alphas and the murineIFN-alphas, and the human IFN-alphas had human IFN-beta. Of the threepreviously defined critical active domains in the Type I IFNs, it isthis domain that exhibits the most divergence with respect to species,and alpha-versus beta-IFNs (Table 1). It is noteworthy that the hybridIFN, IFN-alphaAD(BgI II), exhibits a hydrophilicity plot somewhatdifferent from the human IFN-alphas in this region, yet similar to thatseen for the murine IFNs, specifically MuIFN-alphaCon (FIG. 4). BothMuIFN-alphaCon and IFN-alphaAD(BgI II) have a cysteine residue atposition 86, in contrast with the majority of human IFN-alphas, forwhich there is a tyrosine residue in this position. These data areconsistent with IFN-alphaAD(BgI II) showing demonstrable biologicalactivity on murine cells and support the hypothesis that this region inthe Type I IFNs determines species specificity. Indeed, the hybridIFN-alphaAD(PvuII) resembles the human IFN-alphas in this region (FIG.4) and differs from IFN-alphaAD(BgI II) at just three residue positions,two of which reside in this critical domain: 69 (S/T), 80(T/D) and86(Y/C). IFN-alphaAD(Pvu II) demonstrates considerably reduced antiviralactivity on murine cells compared with IFN-alphaAD(BgI II) yetcomparable activity to IFN-alpha 2a, on human cells.

Sequence homology among the different Type 1 IFNs in conserved regionswould suggest evolutionary significance. It is noteworthy that theamino-and carboxy-terminal domains that have been identified ascritical, are highly conserved among the different molecular subtypes ofType 1 IFNs. Within the 29-35 and 123-140 regions are structural motifsthat are consistent with receptor binding domains: loop structures thatare predominantly hydrophilic and located at the surface of themolecule. Some variation in sequence homology is apparent in the 78-95region. The critical epitopes for Type I IFN receptor recognition areassociated with the residue clusters 29-35 and 130-140, for all speciesof Type I IFNs. These epitopes constitute the receptor binding domainsand are likely located in close spacial proximity to one another in thefolded IFN. The specificity of action of a particular Type I IFN isconferred by the recognition epitope 78-95.

The basis for the specificity of interaction of the 78-95 domain and itsputative cognate binding molecule is unknown. Studies with human growthhormone have shown that receptor binding involves both receptorrecognition, by an epitope on the growth hormone, and dimerization ofreceptors, facilitated through the interaction of a separate epitope onthe growth hormone. By analogy, once an IFN-alpha molecule is bound toits receptor, mediated by the recognition epitopes 29-35 and 130-140,the 78-95 epitope in HuIFN-alpha may interact with another Type 1receptor, effecting dimerization. Using the cross-linking agentdisuccinimidyl suberate for analysis of affinity- labeled cellular IFNbinding components, the inventor and a number of other groups have shownthat IFN-receptor complexes of 80 kDa and 140-160 kDa can be separatedby SDS-PAGE. The molecular weight of the predicted IFN-alpha receptorprotein is 63 kDa and that of the majority of IFN-alphas is 20 kDa,thus, monomer (receptor-IFN) and dimerized-(receptor-IFN-receptor)complexes, may represent the 80 kDa and 40-160 kDa moieties that havebeen detected.

FIG. 5 illustrates a model for the tertiary structure of Type 1 IFNs.This model incorporates a helical bundle core, composed of the fivehelices A to E. The loop structures that constitute the proposedreceptor recognition epitopes, residues 29-35 and 130-140, are shown asheavily shaded, broad lines and are aligned such that they dock in thereceptor groove as shown. The third region implicated in the activeconformation of the Type 1 IFNs, 78-95, is not buried in the receptorgroove and is configured to allow binding to its cognate epitope onanother Type 1 IFN receptor. The shaded areas in helices C and Drepresent residues that are critical for maintaining the correctstructural presentation of the corresponding contiguous recognitionepitopes. In agreement with a number of different models that have beenproposed, the Type I IFNs are comprised predominantly of alpha-helicalbundles that are packed together. The receptor recognition site iscomprised of the AB loop, 29-35 and the D helix and DE loop, 123-140.These are aligned in such a way as to permit the IFN to bind to itsreceptor, in the receptor groove, such that the third epitope, 78-95, isexposed and not buried in the receptor groove. The initial interactionof the IFN molecule with the Type I IFN receptor would account for theabundant, low affinity receptor binding component, extrapolated from theScatchard analyses of the different binding isotherms. The higheraffinity component could be invoked once the IFN molecule is bound toits receptor. The heterogeneity of binding observed for IFN-alpha2a isabsent in IFN-alpha₁ Nδ4, and is explained by the alteration of the29-35 and 78-95 epitopes in IFN-alpha₁ Nδ4, as compared withIFN-alpha2a. This may lead to a reduction in signaling potential of thereceptor-bound IFN and hence a reduction in biological potency.

There is some evidence to suggest that the proliferative state of a cellwill determine whether the high affinity binding component is invoked onIFN-alpha2a binding to its receptor. Non-proliferating cells expressfewer Type I IFN receptors and will not exhibit the characteristicheterogeneity of binding seen with proliferating cells. Interestingly,non-proliferating cells do possess both the 80 kDa and 140-160 kDaIFN-binding complexes. The data indicate that non-proliferating cellslack the high affinity component of IFN-alpha binding, that is notassociated with IFN-receptor dimerization, yet may represent a secondarybinding molecule. A comprehensive binding model, therefore, that wouldaccount for heterogeneity of binding distinct from receptordimerization, would invoke the interaction of the IFN-bound receptorcomplex with a putative secondary binding molecule. The possibility thatother accessory molecules are required for the full complement ofIFN-receptor interactions, is supported by observations of highmolecular weight complexes containing the IFN-alpha-receptor complex.Furthermore, the genetic transfer of the human IFN-alpha receptor intomouse cells, led to transfectants that exhibited a poor sensitivity toselected Type 1 human IFNs. These results infer that the transfectedprotein may not be sufficient for the complete binding activities of theIFNs. Indeed, in the receptor systems described for interleukin-6 andnerve growth factor, accessory proteins are required for the highaffinity binding component of the receptor-ligand interaction. In theabsence of experimental data, it cannot be discounted that the 78-95epitope in Type 1 IFNs may interact with a species-specific secondarybinding molecule. It is intriguing to suggest that the differentialspecificity of action that resides in IFN-alpha and IFN-beta, resultsfrom the specific interaction of the 78-95 region in the two IFNs with acomplementary cognate accessory binding molecule. Moreover, the speciesspecificity observed for the Type 1 IFNs may reside in the recognitionof this species-specific cognate binding molecule, by the specific andvariable 78-95 epitopes amongst the different Type 1 IFN species. Theprecedent for major determinants of specificity of interaction has beenmade with small nuclear ribonucleoproteins and specific RNAs: RNAbinding specificity is conferred by short stretches of variant aminoacid residues in two ribonucleoproteins that otherwise share extensivesequence homology. Certainly, among DNA binding proteins, exchange ofamino acid residues between members of the helix-turn-helix and zincfinger protein families can result in the exchange of DNA bindingspecificity. The nature of the accessory binding molecule that may beassociated with the Type 1 IFN receptor complex remains to be clarified.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CysLeuLysAspArgHisAsp                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspGluSerLeuLeuGluLysPheTyrThrGluLeuTyrGlnGlnLeu                              151015                                                                        AsnAsp                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AsnGluThrIleValGluAsnLeuLeuAlaAsnValTyrHisGlnIle                              151015                                                                        AsnHis                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TyrLeuThrGluLysLysTyrSerProCysAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TyrPheGlnArgIleThrLeuTyrLeuThrGluLysLysTyrSerPro                              151015                                                                        CysAla                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrPheGlnArgIleThrLeuTyr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GluLeuTyrGlnGlnLeuAsnAsp                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 166 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CysAspLeuProGlnThrHisSerLeuGlyAsnArgArgThrLeuIle                              151015                                                                        LeuLeuAlaGlnMetArgArgIleSerProPheSerCysLeuLysAsp                              202530                                                                        ArgHisAspPheGlyPheProGlnGluGluPheAspGlyAsnGlnPhe                              354045                                                                        GlnLysAlaGlnAlaIleSerTyrLeuHisGluMetIleGlnGlnThr                              505560                                                                        PheAsnLeuPheSerThrLysAspSerSerAlaAlaTrpAspGluSer                              65707580                                                                      LeuLeuGluLysPheTyrThrGluLeuTyrGlnGlnLeuAsnAspLeu                              859095                                                                        GluAlaCysTyrIleGlnGluValGlyValGluGluThrProLeuMet                              100105110                                                                     AsnValAspSerIleLeuAlaValArgLysTyrPheGlnArgIleThr                              115120125                                                                     LeuTyrLeuThrGluLysLysTyrSerProCysAlaTrpGluValVal                              130135140                                                                     ArgAlaGluIleMetArgSerPheSerLeuSerThrAsnLeuGlnGlu                              145150155160                                                                  ArgLeuArgArgLysGlu                                                            165                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 166 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysAspLeuProGlnThrHisSerLeuGlySerArgArgThrLeuMet                              151015                                                                        LeuLeuAlaGlnMetArgArgIleSerLeuPheSerCysLeuLysAsp                              202530                                                                        ArgHisAspPheGlyPheProGlnGluGluPheXaaGlyAsnGlnPhe                              354045                                                                        GlnLysAlaGluThrIleProValLeuHisGluMetIleGlnGlnIle                              505560                                                                        PheAsnLeuPheSerThrLysAspSerSerAlaAlaTrpAspGluThr                              65707580                                                                      LeuLeuAspLysPheTyrThrGluLeuTyrGlnGlnLeuAsnAspLeu                              859095                                                                        GluAlaCysTyrIleGlnGlyValGlyValThrGluThrProLeuMet                              100105110                                                                     LysGluAspSerIleLeuAlaValArgLysTyrPheGlnArgIleThr                              115120125                                                                     LeuTyrLeuThrGluLysLysTyrSerProCysAlaTrpGluValVal                              130135140                                                                     ArgAlaGluIleMetArgSerPheSerLeuSerThrAsnLeuGlnGlu                              145150155160                                                                  SerLeuArgSerLysGlu                                                            165                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlnThrHisSerLeuGlySerArgArgThrLeuMetLeuLeuAlaGln                              151015                                                                        MetArgArgIleSerLeuPheSerCysLeuLysAspArgHisAspPhe                              202530                                                                        GlyPheProGlnGluGluPheGlyAsnGlnPheGlnLysAlaGluThr                              354045                                                                        IleProValLeuHisGluMetIleGlnGlnIlePheAsnLeuPheSer                              505560                                                                        ThrLysAspSerSerAlaAlaTrpAspGluThrLeuLeuAspLysPhe                              65707580                                                                      TyrThrGluLeuTyrGlnGlnLeuAsnAspLeuGluAlaCysTyrIle                              859095                                                                        GlnGlyValGlyValThrGluThrProLeuMetLysGluAspSerIle                              100105110                                                                     LeuAlaValArgLysTyrPheGlnArgIleThrLeuTyrLeuThrGlu                              115120125                                                                     LysLysTyrSerProCysAlaTrpGluValValArgAlaGluIleMet                              130135140                                                                     ArgSerPheSerLeuSer                                                            145150                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlnThrHisSerLeuGlySerArgArgThrLeuMetLeuLeuAlaGln                              151015                                                                        MetArgArgIleSerLeuPheSerCysLeuLysAspArgHisAspPhe                              202530                                                                        GlyPheProGlnGluGluPheGlyAsnGlnPheGlnLysAlaGluThr                              354045                                                                        IleProValLeuHisGluMetIleGlnGlnIlePheAsnLeuPheSer                              505560                                                                        ThrLysAspSerSerAlaAlaTrpAspGluThrLeuLeuAspLysPhe                              65707580                                                                      TyrThrGluLeuTyrGlnGlnLeuAsnAspLeuGluAlaCysTyrIle                              859095                                                                        GlnGlyValGlyValThrGluThrProLeuMetLysGluAspSerIle                              100105110                                                                     LeuAlaValArgLysTyrPheGlnArgIleThrLeuTyrLeuThrGlu                              115120125                                                                     LysLysTyrSerProCysAlaTrpGluValValArgAlaGluIleMet                              130135140                                                                     ArgSerPheSerLeuSer                                                            145150                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlnThrHisSerLeuGlySerArgArgThrLeuMetLeuLeuAlaGln                              151015                                                                        MetArgArgIleSerLeuPheSerCysLeuLysAspArgHisAspPhe                              202530                                                                        GlyPheProGlnGluGluPheGlyAsnGlnPheGlnLysAlaGluThr                              354045                                                                        IleProValLeuHisGluMetIleGlnGlnIlePheAsnLeuPheSer                              505560                                                                        ThrLysAspSerSerAlaAlaTrpAspGluThrLeuLeuAspLysPhe                              65707580                                                                      TyrThrGluLeuTyrGlnGlnLeuAsnAspLeuGluAlaCysTyrIle                              859095                                                                        GlnGlyValGlyValThrGluThrProLeuMetLysGluAspSerIle                              100105110                                                                     LeuAlaValArgLysTyrPheGlnArgIleThrLeuTyrLeuThrGlu                              115120125                                                                     LysLysTyrSerProCysAlaTrpGluValValArgAlaGluIleMet                              130135140                                                                     ArgSerPheSerLeuSer                                                            145150                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 165 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CysAspLeuProGluThrHisSerLeuGlySerArgArgThrLeuMet                              151015                                                                        LeuLeuAlaGlnMetArgArgIleSerLeuSerSerCysLeuMetAsp                              202530                                                                        ArgHisAspPheGlyPheProGlnGluGluPheGlyAsnGlnPheGln                              354045                                                                        LysAlaGluThrIleProValLeuHisLeuMetIleGlnGlnIlePhe                              505560                                                                        AsnLeuPheSerThrLysAspSerSerAlaAlaTrpAspGluThrLeu                              65707580                                                                      LeuAspLysPheTyrThrGluLeuTyrGlnGlnLeuAsnAspLeuGlu                              859095                                                                        AlaCysTyrIleGlnGlyValGlyValThrGluThrProLeuMetLys                              100105110                                                                     GluAspSerIleLeuAlaValArgLysTyrPheGlnArgIleThrLeu                              115120125                                                                     TyrLeuThrGluLysLysTyrSerProCysAlaTrpGluValValArg                              130135140                                                                     AlaGluIleMetArgSerPheSerLeuSerThrAsnLeuGlnGluSer                              145150155160                                                                  LeuArgSerLysGlu                                                               165                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 165 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CysAspLeuProGluThrHisSerLeuGlySerArgArgThrLeuMet                              151015                                                                        LeuLeuAlaGlnMetArgArgIleSerLeuPheSerCysAlaLysAla                              202530                                                                        AlaHisAspPheGlyPheProGlnGluGluPheGlyAsnGlnPheGln                              354045                                                                        LysAlaGluThrIleProValLeuHisLeuMetIleGlnGlnIlePhe                              505560                                                                        AsnLeuPheSerThrLysAspSerSerAlaAlaTrpAspGluThrLeu                              65707580                                                                      LeuAspLysPheTyrThrGluLeuTyrGlnGlnLeuAsnAspLeuGlu                              859095                                                                        AlaCysTyrIleGlnGlyValGlyValThrGluThrProLeuMetLys                              100105110                                                                     GluAspSerIleLeuAlaValArgLysTyrPheGlnArgIleThrLeu                              115120125                                                                     TyrLeuThrGluLysLysTyrSerProCysAlaTrpGluValValArg                              130135140                                                                     AlaGluIleMetArgSerPheSerLeuSerThrAsnLeuGlnGluSer                              145150155160                                                                  LeuArgSerLysGlu                                                               165                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 162 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GluThrHisSerLeuAspAsnArgArgThrLeuMetLeuLeuAlaGln                              151015                                                                        MetSerArgIleSerProSerSerCysLeuMetAspArgHisAspPhe                              202530                                                                        GlyPheProGlnGluGluPheAspGlyAsnGlnPheGlnLysAlaPro                              354045                                                                        AlaIleSerValHisLeuGluLeuIleGlnGlnIlePheAsnLeuPhe                              505560                                                                        ThrThrLysAspSerSerAlaAlaTrpAspGluAspLeuLeuAspLys                              65707580                                                                      PheCysThrGluLeuTyrGlnGlnLeuAsnAspLeuGluAlaCysTyr                              859095                                                                        MetGlnGluGluArgValGlyGluThrProLeuMetAsnAlaAspSer                              100105110                                                                     IleLeuAlaValLysLysTyrPheArgArgIleThrLeuTyrLeuThr                              115120125                                                                     GluLysLysTyrSerProCysAlaTrpGluValValArgAlaGluIle                              130135140                                                                     MetArgSerPheSerLeuSerThrAsnLeuGlnGluArgLeuArgArg                              145150155160                                                                  LysGlu                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 166 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetSerTyrAsnLeuLeuGlyPheLeuGlnArgSerSerAsnPheGln                              151015                                                                        CysGlnLysLeuLeuTrpGlnLeuAsnGlyArgLeuGluTyrCysLeu                              202530                                                                        LysAspArgMetAsnPheAspIleProGluGluGluLysGlnLeuGln                              354045                                                                        GlnPheGlnLysGluAspAlaAlaLeuThrIleTyrGluMetLeuGln                              505560                                                                        AsnIlePheAlaIlePheArgGlnAspSerSerSerThrGlyTrpAsn                              65707580                                                                      GluThrIleValGluAsnLeuLeuAlaAsnValValHisGlnAsnHis                              859095                                                                        LeuLysThrValLeuGluGluLysLeuGluLysGluAspPheThrPhe                              100105110                                                                     IleGlyLysLeuMetSerSerLeuHisLeuLysArgTyrTyrGlyArg                              115120125                                                                     IleLeuHisTyrLeuLysAlaLysGluTyrSerHisCysAlaTrpThr                              130135140                                                                     IleValAlaValGluIleLeuArgAsnPheTyrLeuIleAsnArgLeu                              145150155160                                                                  ThrGlyTyrLeuArgAsn                                                            165                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CysAspLeuProGlnThrHisAsnLeuArgAsnLysArgAlaLeuThr                              151015                                                                        LeuLeuValGlnMetArgArgLeuSerProLeuSerCysLeuLysAsp                              202530                                                                        ArgLysAspPheGlyPheProGlnGluLysValAspAlaGlnGlnIle                              354045                                                                        GlnLysAlaGlnAlaIleProValLeuSerGluLeuThrGlnGlnIle                              505560                                                                        LeuAsnIlePheThrSerLysAspSerSerAlaAlaTrpAsnAlaThr                              65707580                                                                      LeuLeuAspSerPheCysAsnAspLeuHisGlnCysLeuAsnAspLeu                              859095                                                                        GlnAlaCysLeuMetGlnGluValGlyValGlnGluProProLeuThr                              100105110                                                                     GlnGluAspSerLeuLeuAlaValArgLysTyrPheHisArgIleThr                              115120125                                                                     ValValLeuArgGluLysLysHisSerProCysAlaTrpGluValVal                              130135140                                                                     ArgAlaGluValValValArgAlaLeuSerSerSerAlaAsnLeuLeu                              145150155160                                                                  AlaArgLeuSerGluGluLysGlu                                                      165                                                                           __________________________________________________________________________

I claim:
 1. A novel IFN-receptor binding peptide having an amino acidsequence of the formula: CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO. 1). 2.A novel IFN-receptor binding peptide having an amino acid sequence ofthe formula:ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP(SEQ. ID NO. 2).
 3. A novel IFN-receptor binding peptide having an aminoacid sequence of the formula:ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS(SEQ. ID NO. 3).
 4. A novel IFN-receptor binding peptide having an aminoacid sequence of the formula:TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA (SEQ. ID NO. 4).
 5. A novelIFN-receptor binding peptide having an amino acid sequence of theformula:TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-HYS-TYR-SER-PRO-CYS-ALA(SEQ. ID NO. 5).
 6. A novel IFN-receptor binding peptide having an aminoacid sequence of the formula: TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR (SEQ. IDNO. 6).
 7. A novel IFN-receptor binding peptide having an amino acidsequence of the formula: GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP (SEQ. ID NO.7).
 8. A polypeptide for use as an interferon-receptor binding peptide,said polypeptide selected from the group of peptides having an aminoacid sequence of the formulae: CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO.1);ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP(SEQ. ID NO. 2);ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS(SEQ. ID NO. 3); TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA (SEQ. IDNO. 4);TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA(SEQ. ID NO. 5); TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR (SEQ. ID NO. 6); andGLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP (SEQ. ID NO. 7).